Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A RNF26 with a C-terminal mCherry tag was expressed with CALR or TGOLN2 (Trans-Golgi network integral membrane protein 2) that has a GFP-tag in HeLa cells. The RNF26 subcellular localization was determined by confocal microscopy. B EBOV-GP 1,2 (GP) with a C-terminal GFP tag was expressed with RNF26-mCherry in HeLa cells, and their co-localization was determined by confocal microscopy. C GP 1,2 proteins from indicated ebolaviruses were expressed with RNF26 in HEK293T cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; RNF26 was detected by anti-FLAG. D A schematic diagram of RNF26 is presented on the top. Five transmembrane (TM1–TM5) domains and a RING-finger are shown. Five RNF26 deletion mutants that target indicated regions and four point-mutation mutants that target each of C395, C399, or C401 are indicated. E EBOV-GP 1,2 (GP) was expressed with RNF26 WT and its mutants in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF26 proteins were detected by anti-FLAG. F EBOV-GP 1,2 (GP) with a HiBiT tag was expressed with RNF26 WT and its mutants in HEK293T cells. RNF26 proteins were immunoprecipitated and their interactions with GP was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26 proteins were detected by anti-FLAG; GFP was detected by its specific antibody. G RNF26-VN and EBOV-GP 1,2 -VC were expressed with CALR that has a C-terminal Blue Fluorescent Protein (BFP) tag in HeLa cells. RNF26 was stained with a red-fluorescent antibody. The subcellular localization of the RNF26-GP complex was determined by confocal microscopy. The scale bar in A , B , and G denotes 5 μm.

Article Snippet: pcDNA3.1-EBOV-GP, pcDNA3.1-EBOV-GP∆MLD, pCMV3-EBOV-VP40, pNL-Luc-△Env, pMSCVneo-CANX-HA, pCMV6-CALR-Myc, and pCAGGS-CANX-HA were reported . pcDNA3.1-GFP-FLAG-HA, pRK5-Ub-His, pCAGGS-PDIA3-Myc, pCAGGS-PDIA3-HA, pcDNA3.1 vectors expressing EBOV-GP, GP△MLD, sGP or ssGP with a N-terminal HiBiT tag, pCAGGS-MARV-GP with a C-terminal HiBiT tag, pcDNA3.1 vectors expressing GPs from BDBV, RETV, SBOV, or TAFV with a HA-FLAG-tag, and vectors expressing HIV-1 Env, IAV-H5 HA, and VSV-G were reported . pCAGGS-MARCH8-HA and its W114A mutant were reported . pEGFPC1 was from Takara. pCMV6-CANX-Myc-FLAG was constructed from pCAGGS-CANX-HA by PCR and ASiSI/MluI digestion. pCAGGS vectors expressing MERS-CoV-S, SRAS-CoV-S, and SARS-CoV2-S with a FLAG-tag were constructed by PCR and EcoRI/XhoI digestion. pEGFPN1 vectors expressing CALR, TGLON2, and EBOV-GP were constructed by PCR and XhoI/BspEI digestion. pcDNA3.1-EBOV-GP-HA was constructed by PCR and NheI/HindIII digestion. pmBFPN1-CALR was constructed from pEGFPN1-CALR by replacing EGFP with mBFP via AgeI/NotI digestion. pRK5-HA-Ub-WT was obtained from Ted Dawson via Addgene (#17608). pCMV6-RNF26-Myc-FLAG, pCMV6-RNF185-Myc-FLAG, and pcDNA3.1-TRIM25-FLAG were ordered from CoME, ORIGENE, or JINSIRUI. pCAGGS-RNF26-mCherry was constructed from pCAGGS-ACE2-mCherry by PCR and EcoRI digestion followed by homologous recombination (Vazyme, C113-01). pcDNA3.1-RNF26-VN-HA was constructed from pcDNA3.1-ACE2-VN-HA by PCR and XhoI/BspEI digestion. pcDNA3.1-FLAG-EBOV-GP-FLAG-VC was constructed from pcDNA3.1-ACE2-FLAG-VC by PCR and XhoI/BspEI digestion.

Techniques: Membrane, Confocal Microscopy, Expressing, Mutagenesis, Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A Three RNF26 -KO cell lines (1-E4, 3-B8, 3-F10) were generated from HEK293T cells using CRISPR/Cas9. RNF26 expression in these cells was determined by WB using its specific antibody. B EBOV and RSTV GP 1,2 proteins were expressed in HEK293T WT and RNF26 -KO (1-E4) cells. GP expression was detected by WB using anti-EBOV-GP. C EBOV-GP 1,2 (GP) was expressed with PDIA3, CALR, or CANX in HEK293T WT and RNF26 -KO (1-E4) cells. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA. D RNF26 was expressed with GFP, PDIA3, CALR, or CANX in HEK293T cells. RNF26 was co-immunoprecipitated (IP) from cell lysate (Input) and its interactions with these proteins was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; RNF26 was detected by anti-FLAG; GFP was detected by its specific antibody. E GP was expressed with His-tagged ubiquitin (Ub) and PDIA3, CALR, CANX, and RNF26 or its catalytically inactive mutant (3C/3S) in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; Ub was detected by anti-His.

Article Snippet: pcDNA3.1-EBOV-GP, pcDNA3.1-EBOV-GP∆MLD, pCMV3-EBOV-VP40, pNL-Luc-△Env, pMSCVneo-CANX-HA, pCMV6-CALR-Myc, and pCAGGS-CANX-HA were reported . pcDNA3.1-GFP-FLAG-HA, pRK5-Ub-His, pCAGGS-PDIA3-Myc, pCAGGS-PDIA3-HA, pcDNA3.1 vectors expressing EBOV-GP, GP△MLD, sGP or ssGP with a N-terminal HiBiT tag, pCAGGS-MARV-GP with a C-terminal HiBiT tag, pcDNA3.1 vectors expressing GPs from BDBV, RETV, SBOV, or TAFV with a HA-FLAG-tag, and vectors expressing HIV-1 Env, IAV-H5 HA, and VSV-G were reported . pCAGGS-MARCH8-HA and its W114A mutant were reported . pEGFPC1 was from Takara. pCMV6-CANX-Myc-FLAG was constructed from pCAGGS-CANX-HA by PCR and ASiSI/MluI digestion. pCAGGS vectors expressing MERS-CoV-S, SRAS-CoV-S, and SARS-CoV2-S with a FLAG-tag were constructed by PCR and EcoRI/XhoI digestion. pEGFPN1 vectors expressing CALR, TGLON2, and EBOV-GP were constructed by PCR and XhoI/BspEI digestion. pcDNA3.1-EBOV-GP-HA was constructed by PCR and NheI/HindIII digestion. pmBFPN1-CALR was constructed from pEGFPN1-CALR by replacing EGFP with mBFP via AgeI/NotI digestion. pRK5-HA-Ub-WT was obtained from Ted Dawson via Addgene (#17608). pCMV6-RNF26-Myc-FLAG, pCMV6-RNF185-Myc-FLAG, and pcDNA3.1-TRIM25-FLAG were ordered from CoME, ORIGENE, or JINSIRUI. pCAGGS-RNF26-mCherry was constructed from pCAGGS-ACE2-mCherry by PCR and EcoRI digestion followed by homologous recombination (Vazyme, C113-01). pcDNA3.1-RNF26-VN-HA was constructed from pcDNA3.1-ACE2-VN-HA by PCR and XhoI/BspEI digestion. pcDNA3.1-FLAG-EBOV-GP-FLAG-VC was constructed from pcDNA3.1-ACE2-FLAG-VC by PCR and XhoI/BspEI digestion.

Techniques: Generated, CRISPR, Expressing, Immunoprecipitation, Mutagenesis

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP was expressed with His-tagged Ub in the presence of indicated E3 Ub ligases or their catalytically inactive mutants in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP was expressed with indicated proteins in HEK293T cells. Protein expression was determined by WB. GP, RNF26, RNF185, TRIM25, and MARCH8 were detected as in A ; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA. C GP was expressed with RNF185 or RNF26 and treated with indicated inhibitors as we did previously. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF26 and RNF185 proteins were detected by anti-FLAG. D A schematic diagram of RNF185 is presented. Two transmembrane (TM1, TM2) domains and a RING-finger are shown. Indicated RNF26 mutants were constructed. E GP was expressed with RNF185 and its mutants in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG. F GP was expressed with GFP, RNF185, and indicated RNF185 mutants in HEK293T cells. GFP and RNF185 proteins were immunoprecipitated and their interaction with GP was analyzed by WB. GP was detected by anti-EBOV-GP; GFP and RNF185 proteins were detected by anti-FLAG. G GP was expressed with Ub with a His-tag and RNF185 or its indicated mutants in HEK293T cells. GP and RNF185 proteins were immunoprecipitated and GP polyubiquitination was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG; Ub was detected by anti-His.

Article Snippet: pcDNA3.1-EBOV-GP, pcDNA3.1-EBOV-GP∆MLD, pCMV3-EBOV-VP40, pNL-Luc-△Env, pMSCVneo-CANX-HA, pCMV6-CALR-Myc, and pCAGGS-CANX-HA were reported . pcDNA3.1-GFP-FLAG-HA, pRK5-Ub-His, pCAGGS-PDIA3-Myc, pCAGGS-PDIA3-HA, pcDNA3.1 vectors expressing EBOV-GP, GP△MLD, sGP or ssGP with a N-terminal HiBiT tag, pCAGGS-MARV-GP with a C-terminal HiBiT tag, pcDNA3.1 vectors expressing GPs from BDBV, RETV, SBOV, or TAFV with a HA-FLAG-tag, and vectors expressing HIV-1 Env, IAV-H5 HA, and VSV-G were reported . pCAGGS-MARCH8-HA and its W114A mutant were reported . pEGFPC1 was from Takara. pCMV6-CANX-Myc-FLAG was constructed from pCAGGS-CANX-HA by PCR and ASiSI/MluI digestion. pCAGGS vectors expressing MERS-CoV-S, SRAS-CoV-S, and SARS-CoV2-S with a FLAG-tag were constructed by PCR and EcoRI/XhoI digestion. pEGFPN1 vectors expressing CALR, TGLON2, and EBOV-GP were constructed by PCR and XhoI/BspEI digestion. pcDNA3.1-EBOV-GP-HA was constructed by PCR and NheI/HindIII digestion. pmBFPN1-CALR was constructed from pEGFPN1-CALR by replacing EGFP with mBFP via AgeI/NotI digestion. pRK5-HA-Ub-WT was obtained from Ted Dawson via Addgene (#17608). pCMV6-RNF26-Myc-FLAG, pCMV6-RNF185-Myc-FLAG, and pcDNA3.1-TRIM25-FLAG were ordered from CoME, ORIGENE, or JINSIRUI. pCAGGS-RNF26-mCherry was constructed from pCAGGS-ACE2-mCherry by PCR and EcoRI digestion followed by homologous recombination (Vazyme, C113-01). pcDNA3.1-RNF26-VN-HA was constructed from pcDNA3.1-ACE2-VN-HA by PCR and XhoI/BspEI digestion. pcDNA3.1-FLAG-EBOV-GP-FLAG-VC was constructed from pcDNA3.1-ACE2-FLAG-VC by PCR and XhoI/BspEI digestion.

Techniques: Immunoprecipitation, Expressing, Construct

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP and its mutants K673A or ∆CT were expressed with indicated E3 Ub ligases in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP and its two mutants were expressed with His-tagged Ub in the presence of PDIA3, CALR, or CANX in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; Ub was detected by anti-His. C GP and its two mutants were expressed with indicated E3 ubiquitin ligases or ER proteins in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; MARCH8 and CANX were detected by anti-HA; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; PDIA3 and CALR were detected by anti-Myc. D RNF185 was expressed with a control (Ctrl) or RNF185 -specific siRNAs in HEK293 cells. RNF185 expression was determined by WB using anti-FLAG. E GP was expressed with PDIA3, CALR, or CANX in the presence of Ctrl or RNF185 -specific siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP. F GP was expressed with CALR, CANX, or PDIA3, and indicated siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP.

Article Snippet: pcDNA3.1-EBOV-GP, pcDNA3.1-EBOV-GP∆MLD, pCMV3-EBOV-VP40, pNL-Luc-△Env, pMSCVneo-CANX-HA, pCMV6-CALR-Myc, and pCAGGS-CANX-HA were reported . pcDNA3.1-GFP-FLAG-HA, pRK5-Ub-His, pCAGGS-PDIA3-Myc, pCAGGS-PDIA3-HA, pcDNA3.1 vectors expressing EBOV-GP, GP△MLD, sGP or ssGP with a N-terminal HiBiT tag, pCAGGS-MARV-GP with a C-terminal HiBiT tag, pcDNA3.1 vectors expressing GPs from BDBV, RETV, SBOV, or TAFV with a HA-FLAG-tag, and vectors expressing HIV-1 Env, IAV-H5 HA, and VSV-G were reported . pCAGGS-MARCH8-HA and its W114A mutant were reported . pEGFPC1 was from Takara. pCMV6-CANX-Myc-FLAG was constructed from pCAGGS-CANX-HA by PCR and ASiSI/MluI digestion. pCAGGS vectors expressing MERS-CoV-S, SRAS-CoV-S, and SARS-CoV2-S with a FLAG-tag were constructed by PCR and EcoRI/XhoI digestion. pEGFPN1 vectors expressing CALR, TGLON2, and EBOV-GP were constructed by PCR and XhoI/BspEI digestion. pcDNA3.1-EBOV-GP-HA was constructed by PCR and NheI/HindIII digestion. pmBFPN1-CALR was constructed from pEGFPN1-CALR by replacing EGFP with mBFP via AgeI/NotI digestion. pRK5-HA-Ub-WT was obtained from Ted Dawson via Addgene (#17608). pCMV6-RNF26-Myc-FLAG, pCMV6-RNF185-Myc-FLAG, and pcDNA3.1-TRIM25-FLAG were ordered from CoME, ORIGENE, or JINSIRUI. pCAGGS-RNF26-mCherry was constructed from pCAGGS-ACE2-mCherry by PCR and EcoRI digestion followed by homologous recombination (Vazyme, C113-01). pcDNA3.1-RNF26-VN-HA was constructed from pcDNA3.1-ACE2-VN-HA by PCR and XhoI/BspEI digestion. pcDNA3.1-FLAG-EBOV-GP-FLAG-VC was constructed from pcDNA3.1-ACE2-FLAG-VC by PCR and XhoI/BspEI digestion.

Techniques: Immunoprecipitation, Expressing

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: ACE2 orthologs of diverse species mediate SARS-CoV-2 infection. (A) 293T cells transfected with plasmids expressing 10 ACE2 orthologs or an empty vector and then infected by icSARS-CoV-2-mNG at a multiplicity of infection of 1 for 48 h. Images were captured by using a Zeiss LSM 880 laser scanning microscope. (B) Quantification of infection by flow cytometry. Cells from panel A were fixed in 4% paraformaldehyde, and mNG-positive cells were then quantified by flow cytometric analysis. SSC, side scatter. (C) The expression of ACE2 ortholog plasmids in 293T cells was detected using a mouse anti-V5 tag monoclonal antibody targeting the C-terminal V5 tag (in red). Green, anti-β-actin antibody. (D) Representative confocal images of Caco-2 and Calu-3 cells infected with icSARS-CoV-2-mNG at a multiplicity of infection of 1 for 48 h.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Laser-Scanning Microscopy, Flow Cytometry

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: PRRA-led proteolytic cleavage of SARS-CoV-2 spike protein and effect on fusion. (A) Organization of SARS-CoV-2 spike protein and sequence alignment at the S1/S2 site with RaTG13 and pangolin GX S proteins. SP, signal peptide; NTD, N-terminal domain; FP, fusion peptide; TM, transmembrane; CT, C terminus. (B) 293T cells transfected with SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 spike (RaTG13 S), an insertion mutant containing PRRA (RaTG13 S+PRRA), and the pangolin GX spike protein (Pangolin GX S) (lanes 3 to 7) and SARS-CoV-2 S-infected Vero E6, Calu-3, and Caco-2 cells (lanes 8 to 10). A 5-min exposure of this part of the gel is included. Red, anti-S antibody; green, anti-β-actin antibody. (C) Cell-cell fusion mediated by CoV S proteins. 293T cells expressing Stop‐Luc, ACE2, and/or ACE2/TMPRSS2 (acceptor cells) were mixed at a 1:1 ratio with donor cells expressing Cre, CoV S, or both to initiate cell‐cell fusion. Data are presented as means ± standard errors of the means (SEM). (D) 293T cells or 293T-hACE2 cells were infected by pseudovirus bearing SARS-CoV-2 S. Bald viruses without any viral envelope or VSV-Gpp were included as negative and positive controls. (E) Entry of MLV pseudoviruses bearing SARS-CoV-2 S or SARS-CoV-2 S ΔPRRA in Vero cells. **, P < 0.001; ***, P < 0.0001. RLU, relative luciferase units.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Sequencing, Transfection, Mutagenesis, Infection, Expressing, Luciferase

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The PRRA insert at the S1/S2 site distinctly modulates cell susceptibility to SARS-CoV-2 pseudovirions. (A to C) Entry of HIV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 S, RaTG13 S+PRRA, and pangolin GX S into 293T cells transiently expressing ACE2 (A), Calu-3 cells (B), and Caco-2 cells (C). (D) Western blot analysis of pseudovirions bearing SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 S, RaTG13 S+PRRA, and pangolin GX S. Red, anti-p24 antibody; green, antispike antibody. (E) Infection by pseudovirions harboring the indicated viral glycoproteins in 293T cells transfected with ACE2 alone or in combination with TMPRSS2. Data are presented as means ± SEM. *, P < 0.01.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The novel S1/S2 site alters the dependence of RaTG13 pseudovirus on ACE2 orthologs. Shown are the infectivities of pseudovirions harboring the indicated viral spike proteins on 293T cells transiently transfected with Chinese hamster ACE2 and bovine ACE2 (A), Syrian hamster ACE2 and human ACE2 (B), ferret ACE2 and pig ACE2 (C), Agm ACE2 and mouse ACE2 (D), and horseshoe bat ACE2 and Malayan pangolin ACE2 (E). Data are presented as means ± SEM. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001. Numbers above the asterisks denote the fold changes. (F) Binding of pseudovirions carrying SARS-CoV-2 S, RaTG13 S, and RaTG13 S+PRRA to full-length recombinant human and mouse ACE2 was quantified by qPCR (see Materials and Methods). Binding relative to that of bald virus is plotted.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Transfection, Binding Assay, Recombinant, Virus

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The novel S1/S2 site alters the dependence of RaTG13 pseudovirus on ACE2 orthologs in the presence of TMPRSS2. 293T cells expressing each ACE2 ortholog with TMPRSS2 were infected by pseudoviruses harboring SARS-CoV-2 S (A), SARS-CoV-2 S ΔPRRA (B), RaTG13 S (C), RaTG13 S+PRRA (D), pangolin S (E), and VSV-G (F). Luciferase activities in cell lysates were determined at 48 h postinfection. Data are presented as means ± SEM. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Expressing, Infection, Luciferase

Journal: Cell Reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Binding affinity and cryo-EM structure of the Omicron BA.2 S protein-human ACE2 complex (A) Surface plasmon resonance experiments measuring dimeric human ACE2 (hACE2) binding to immobilized wild-type (WT), BA.1, and BA.2 RBDs, performed in technical triplicates. Summary data are shown at the top with representative surface plasmon resonance (SPR)-binding curves (colored solid line), and fitted 1:1 binding models (black dashed line) are shown on bottom. (B) As in (A) but measuring WT, BA.1, and BA.2 RBDs binding to immobilized dimeric hACE2, performed in at least technical quadruplicates. (C) As in (A) but measuring WT, BA.1, and BA.2 ectodomains binding to immobilized dimeric hACE2, performed in at least technical duplicates. The WT and BA.1 data in (C) were previously reported. Pairwise statistical significance test was performed using a one-way ANOVA test ( ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001, ns, not significant). (D) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD in complex with hACE2 at 2.8 Å. (E) Aligned atomic models of hACE2 bound to BA.1 and BA.2 RBDs. The BA.1 RBD (PDB: 7T9L ) and complexed hACE2 atomic models are shown in magenta and dark blue, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light blue, respectively. (F) Atomic model of the BA.1 S protein-hACE2 complex, focused on residue S496. The hydrogen bonding interaction between BA.1 S protein residue S496 and hACE2 residue K353 is indicated by an orange dashed line. (G) As in (F) but for the BA.2 S protein-hACE2 complex, focused on residue G496.

Article Snippet: The mouse ACE2 gene was ordered from Addgene (Plasmid #158087) and amino acid residues 1-615 were amplified and inserted into pcDNA3.1 with a C-terminal HRV 3C site, 8x histidine tag and twin strep tag.

Techniques: Binding Assay, Cryo-EM Sample Prep, SPR Assay, Residue

Journal: Cell Reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Cryo-EM structure of the Omicron BA.2 S protein-mouse ACE2 complex (A) Cryo-EM density map of BA.2 S protein in complex with mouse ACE2 at 2.5 Å. Mouse ACE2 is shown in green, and protomers of the BA.2 S protein are shown in shades of purple. (B) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD-mouse ACE2 (mACE2) complex at 2.7 Å. (C) Aligned atomic models of the BA.1 and BA.2 RBD-mACE2 complexes. The BA.1 RBD and complexed hACE2 atomic models are shown in magenta and dark green, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light green, respectively. (D) Atomic model of the BA.2 RBD-mACE2 complex, focused on residues Y501 and H505. (E) As in (D) but focused on residue R493. (F) Atomic model of the WT RBD-hACE2, focused on residues N501 and Y505. (G) As in (H) but focused on residue Q493. (H) Atomic model of mACE2 from the perspective of a binding RBD. Black labels are mACE2 residues, and gray labels denote the interacting residues in a bound RBD. Gold labels denote the interacting residues in a bound RBD that are mutated in the BA.1 and BA.2 Omicron sub-lineages.

Article Snippet: The mouse ACE2 gene was ordered from Addgene (Plasmid #158087) and amino acid residues 1-615 were amplified and inserted into pcDNA3.1 with a C-terminal HRV 3C site, 8x histidine tag and twin strep tag.

Techniques: Cryo-EM Sample Prep, Residue, Binding Assay

Journal: Cell Reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet:

Article Snippet: The mouse ACE2 gene was ordered from Addgene (Plasmid #158087) and amino acid residues 1-615 were amplified and inserted into pcDNA3.1 with a C-terminal HRV 3C site, 8x histidine tag and twin strep tag.

Techniques: Recombinant, Software

Journal: Cell reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein.

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Figure 2. Binding affinity and cryo-EM structure of the Omicron BA.2 S protein-human ACE2 complex (A) Surface plasmon resonance experiments measuring dimeric human ACE2 (hACE2) binding to immobilized wild-type (WT), BA.1, and BA.2 RBDs, performed in technical triplicates. Summary data are shown at the top with representative surface plasmon resonance (SPR)-binding curves (colored solid line), and fitted 1:1 binding models (black dashed line) are shown on bottom. (B) As in (A) but measuring WT, BA.1, and BA.2 RBDs binding to immobilized dimeric hACE2, performed in at least technical quadruplicates. (C) As in (A) but measuring WT, BA.1, and BA.2 ectodomains binding to immobilized dimeric hACE2, performed in at least technical duplicates. The WT and BA.1 data in (C) were previously reported.9 Pairwise statistical significance test was performed using a one-way ANOVA test (*p % 0.05; **p % 0.01; ***p % 0.001; ****p % 0.0001, ns, not significant). (D) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD in complex with hACE2 at 2.8 A˚ . (E) Aligned atomic models of hACE2 bound to BA.1 and BA.2 RBDs. The BA.1 RBD (PDB: 7T9L) and complexed hACE2 atomic models are shown in magenta and dark blue, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light blue, respectively. (F) Atomic model of the BA.1 S protein-hACE2 complex, focused on residue S496. The hydrogen bonding interaction between BA.1 S protein residue S496 and hACE2 residue K353 is indicated by an orange dashed line. (G) As in (F) but for the BA.2 S protein-hACE2 complex, focused on residue G496.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies VH-FC ab8 (Li et al.)18 N/A Fab S309 (Pinto et al.)20 N/A Fab S2M11 (Tortorici et al.)19 N/A Fab 4-8 (Liu et al.)21 N/A Fab 4A8 (Chi et al.)22 N/A Fab DH1052 (Li et al.)23 N/A Goat anti-human IgG Jackson ImmunoReserach Cat. # 109-035-088; RRID: AB_2337584 Mouse anti-his tag antibody abcam Cat# ab18184; RRID: AB_444306 Chemicals, peptides, and recombinant proteins Ace2 (18-615) New England Biolabs Cat. # 73775S BA.1 RBD Sino Biological Cat# 40592-V08H121 Critical commercial assays Pierce 1-Step Ultra Substrate Solution ThermoFisher Cat. # 34028 Deposited data S(BA.2) This paper Global refinement: EMDB 27523, PDB: 8DM1 Focus refinement: EMDB 27524, PDB: 8DM2 S(BA.2)+hACE2 This paper Global refinement: EMDB 27527, PDB: 8DM5 Focus refinement: EMDB 27528, PDB: 8DM6 S(BA.2)+mACE2 This paper Global refinement: EMDB 27529, PDB: 8DM7 Focus refinement: EMDB 27530, PDB: 8DM8 S(BA.1)+mACE2 This paper Global refinement: EMDB 27531, PDB: 8DM9 Focus refinement: EMDB 27532, PDB: 8DMA S(BA.2)+4A8 This paper Global refinement: EMDB 27525, PDB: 8DM3 Focus refinement: EMDB 27526, PDB: 8DM4 Experimental models: Cell lines Expi293F ThermoFisher Cat# A14527 Recombinant DNA pcDNA3.1 HexaPro BA.1 Mannar et al.9 N/A pcDNA3.1 BA.1 NTD This paper N/A pcDNA3.1 HexaPro BA.2 This paper N/A pcDNA3.1 BA.2 NTD This paper N/A pcDNA3.1 BA.2 RBD This paper N/A pcDNA3.1 hACE2 (1-615) Mannar et al.9 N/A pcDNA3.1 mACE2 (1-615) This paper N/A pCSCG hACE2-FC Yang et al.39 Addgene: 164222 pcDNA3.1 Fab S309 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S309 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab 4-8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4-8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab DH1052 Light Chain This paper N/A (Continued on next page) 10 Cell Reports 42, 111964, January 31, 2023

Techniques: Binding Assay, Cryo-EM Sample Prep, SPR Assay, Residue

Journal: Cell reports

Article Title: Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein.

doi: 10.1016/j.celrep.2022.111964

Figure Lengend Snippet: Figure 3. Cryo-EM structure of the Omicron BA.2 S protein-mouse ACE2 complex (A) Cryo-EM density map of BA.2 S protein in complex with mouse ACE2 at 2.5 A˚ . Mouse ACE2 is shown in green, and protomers of the BA.2 S protein are shown in shades of purple. (B) Focus-refined cryo-EM density map and fitted atomic model of the BA.2 RBD-mouse ACE2 (mACE2) complex at 2.7 A˚ . (C) Aligned atomic models of the BA.1 and BA.2 RBD-mACE2 complexes. The BA.1 RBD and complexed hACE2 atomic models are shown in magenta and dark green, respectively. The BA.2 RBD and complexed hACE2 atomic models are shown in purple and light green, respectively. (D) Atomic model of the BA.2 RBD-mACE2 complex, focused on residues Y501 and H505. (E) As in (D) but focused on residue R493. (F) Atomic model of the WT RBD-hACE2, focused on residues N501 and Y505. (G) As in (H) but focused on residue Q493. (H) Atomic model of mACE2 from the perspective of a binding RBD. Black labels are mACE2 residues, and gray labels denote the interacting residues in a bound RBD. Gold labels denote the interacting residues in a bound RBD that are mutated in the BA.1 and BA.2 Omicron sub-lineages.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies VH-FC ab8 (Li et al.)18 N/A Fab S309 (Pinto et al.)20 N/A Fab S2M11 (Tortorici et al.)19 N/A Fab 4-8 (Liu et al.)21 N/A Fab 4A8 (Chi et al.)22 N/A Fab DH1052 (Li et al.)23 N/A Goat anti-human IgG Jackson ImmunoReserach Cat. # 109-035-088; RRID: AB_2337584 Mouse anti-his tag antibody abcam Cat# ab18184; RRID: AB_444306 Chemicals, peptides, and recombinant proteins Ace2 (18-615) New England Biolabs Cat. # 73775S BA.1 RBD Sino Biological Cat# 40592-V08H121 Critical commercial assays Pierce 1-Step Ultra Substrate Solution ThermoFisher Cat. # 34028 Deposited data S(BA.2) This paper Global refinement: EMDB 27523, PDB: 8DM1 Focus refinement: EMDB 27524, PDB: 8DM2 S(BA.2)+hACE2 This paper Global refinement: EMDB 27527, PDB: 8DM5 Focus refinement: EMDB 27528, PDB: 8DM6 S(BA.2)+mACE2 This paper Global refinement: EMDB 27529, PDB: 8DM7 Focus refinement: EMDB 27530, PDB: 8DM8 S(BA.1)+mACE2 This paper Global refinement: EMDB 27531, PDB: 8DM9 Focus refinement: EMDB 27532, PDB: 8DMA S(BA.2)+4A8 This paper Global refinement: EMDB 27525, PDB: 8DM3 Focus refinement: EMDB 27526, PDB: 8DM4 Experimental models: Cell lines Expi293F ThermoFisher Cat# A14527 Recombinant DNA pcDNA3.1 HexaPro BA.1 Mannar et al.9 N/A pcDNA3.1 BA.1 NTD This paper N/A pcDNA3.1 HexaPro BA.2 This paper N/A pcDNA3.1 BA.2 NTD This paper N/A pcDNA3.1 BA.2 RBD This paper N/A pcDNA3.1 hACE2 (1-615) Mannar et al.9 N/A pcDNA3.1 mACE2 (1-615) This paper N/A pCSCG hACE2-FC Yang et al.39 Addgene: 164222 pcDNA3.1 Fab S309 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S309 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Light Chain Mannar et al.30 N/A pcDNA3.1 Fab S2M11 Heavy Chain Mannar et al.30 N/A pcDNA3.1 Fab 4-8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4-8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Light Chain Saville et al.40 N/A pcDNA3.1 Fab 4A8 Heavy Chain Saville et al.40 N/A pcDNA3.1 Fab DH1052 Light Chain This paper N/A (Continued on next page) 10 Cell Reports 42, 111964, January 31, 2023

Techniques: Cryo-EM Sample Prep, Residue, Binding Assay